Mouse testosterone 16Alpha- and 15Alpha-hydroxylases consist of male and female-specific isozymes. Sex-dependent differences of the 16Alpha- and 15Alpha-hydroxylase activities in microsomes are a sum of the differential expression of the isozymes in males and females. We have purified the male- and female-specific isozymes of 16Alpha-hydroxylase and the female-specific 15Alpha-hydroxylase and prepared specific inhibitory antibodies to the isozymes. The expression of 16Alpha- and/or 15Alpha-hydroxylase activities in microsomes differs among inbred strains of mice; the 16Alpha-hydroxylase is a male-predominant activity in 129/J but not in BALB/cJ mice. The 15Alpha-hydroxylase activity is 10 times as high in renal microsomes of 129/J females than in those of BALB/cJ females. With the use of specific inhibitory antibodies to the isozymes, the genetic basis of strain differences in the hydroxylase activities was studied with offspring of 129CF1(129/J(female)XBALB/cJ), C129F1(BALB/cJ(female)XBALB/cJ), F2(129CF1X129CF1), 129CF1(female)X129/J, and 129CF1(female)XBALB/cJ mice. The low rate of 16Alpha-hydroxylase activity is due to a repression of the female-specific isozyme in 129/J females, and the repression is inherited as an autosomal recessive trait which is regulated by a single locus (Rip locus). A similar study was carried out for regulation of 15Alpha-hydroxylase activity in renal microsomes. Its results demonstrated that the female-specific isozyme of 15alpha-hydroxylase is expressed 10 times more in 129/J than in BALB/cJ females, and that the higher level of expression is inherited as an autosomal dominant trait which is also regulated by a single locus (RSH locus). The cDNA clones encoding these isozymes of testosterone hydroxylases have been isolated. A genomic DNA library of 129/J was constructed. The genomic DNA clones for the isozyme genes have been isolated from BALB/cJ and C57BL/6J as well as 129/J mice of genomic DNA libraries. By characterization of the cDNA and genomic DNA clones, we intend to elucidate the molecular mechanisms of genetic regulation of the sex-dependent expression of these hydroxylase isozymes in mice.